Cancer is one of the leading causes of death in the United States. The majority of fatal human cancers are carcinomas, with no known viral involvement. Some cancers can be induced by environmental exposure to various carcinogens, specifically including the alkylating agents such as beta-propiolactone, methyl-methanesulfonate and methyl-nitrosourea. Induced tumors in animal model systems have allowed the identification of some of the genetic mechanisms of carcinogenesis. During the past few years, numerous oncogenes have been identified on the basis of their ability to transform NIH 3T3 cells when inserted by DNA-mediated transfection. Approximately 20% of human tumors contain such transforming genes. The proposed research is intended to induce tumors in rats through an environmentally significant mechanism, the inhalation of direct-acting carcinogens, and then to isolate the activated oncogenes through transfection schemes that are not as restrictive as those employed in the past. These methods include, in addition to the usual transfection of tumor DNA into NIH 3T3 cells in vitro and picking transformed foci, the cotransfection of tumor DNA with either myc or ras in an aph expression vector into primary mouse fibroblasts. Cotransfected cells will be selected on the basis of their resistance to the antibiotic G418, and injected into nude mice. Tumors formed by this method will be used as the source of DNA for secondary cotransfections. DNA extracted from the secondary tumors will be used in Southern blot analysis of rat-derived sequences, and the creation of recombinant DNA genomic libraries. These libraries will be screened for rat-derived sequences, and clones thus identified will be examined to determine whether they contain transforming genes. These methods should allow the isolation of one or more transforming gene from each tumor tested. The isolated genes will be compared with their normal counterparts by restriction mapping, hybridization studies, S1 nuclease mapping of heteroduplexed DNAs, and sequencing. Such comparisons will allow the determination of the exact nature of the activating mutation. The gene activated, the nature of the activating mutation, and the properties of the activated oncogene will be correlated with the carcinogen used. Information gained in this study will further our knowledge about how normal cells control their growth, and how carcinogenesis can subvert these controls. Such knowledge may enable the design of specific, direct and effective treatments of cancers.
